Functional Role of Pseudouridine on Zika Virus Gene Expression

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Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus, that during the 2015-2016 outbreak in the Americas was associated with severe and devastating developmental abnormalities, including microcephaly, in babies born from infected women. The single-stranded positive-sense RNA genome of ZIKV directs translation of viral proteins, functions as a template for replication of the genome and is packaged into new virus particles. These functions are in part regulated by specific RNA structures within the untranslated regions of the RNA, and via interactions with viral and cellular proteins. More recently RNA modifications have been shown to significantly affect the virus infectious cycle. Although more than 170 different RNA modifications are known to decorate natural RNAs, only N6-methyladenosine has been reported to affect ZIKV gene expression. The role of other RNA modifications on ZIKV RNA is presently unknown. Using biotinylated antisense oligonucleotides to affinity isolate virus RNA from cells and virions, and mass spectrometry to identify RNA modifications, we discovered that more than 30 different RNA modifications are present on ZIKV RNA. In this application we focus on pseudouridine (Y), an abundant but understudied RNA modification. The incorporation of Y on cellular RNAs affects the folding, stability, and translation of RNA, and RNA-RNA and RNA-protein interactions. We propose that Y on the ZIKV RNA genome promotes virus translation and replication. In this application we propose using a next generation sequencing approach combined with chemical modification of pseudouridine to identify specific Y sites on ZIKV genome (Aim 1). Next, we will undertake RNAi depletion and overexpression of cellular pseudouridine synthase proteins to identify the host enzymes responsible for installing pseudouridine on ZIKV RNA (Aim 2). Last, we will use mutagenesis and modulation of the pseudouridine synthase enzymes to elucidate if pseudouridine regulates ZIKV translation and/or replication (Aim 3). Moreover, these studies will be undertaken in the context of virus infection in mammalian and mosquito cells, two hosts biologically relevant to the virus life cycle, which could inform the role of RNA modifications in virus transmission and host adaptation. We expect that this research will advance our knowledge of RNA modifications which have been shown to be novel and important regulators of viral gene expression and improve our understanding of the molecular biology of ZIKV. This research will therefore provide critical information on a virus with significant impacts on human health and that currently lacks therapeutic options.